Projectsummary ThelongtermgoalofthisapplicationistostudyHIV-1reservoirsthatpersistinCNSandtounderstand theroleofillicitdrugsinestablishingandreactivatingthelatentreservoirs.Persistenceoflatentlyinfectedcells inCNSposesamajorbarrierforHIV-1eradication.Currentstrategiestoeliminatethelatentreservoirsinclude a ?shock and kill? therapy and is aimed at peripheral blood, which constitutes only 1% of the total reservoirs. Whether it is possible to envision similar strategies for the eradication of HIV-infected CNS cells is currently unknown. In addition to the lack of knowledge about latency in CNS HIV+ cells and the effects of latency reversingagents,illicitdrugscommonwithintheHIV-infectedpopulationsconstituteafurthercomplexity.Many illicitdrugsareknowntostimulateHIV-1replication.Sincethecurrentmethodtomeasuretheperipheralblood HIVreservoirisnotapplicabletosolidtissuesortocellsthatreplicatepoorlysuchasmacrophages,microglia and astrocytes found in the brain. We have developed a novel, Single cell-single molecule, Multiplex, Immunofluorescence (IF) and RNA FISH-based Assay (SMIRA) to detect cells in which HIV-1 is actively replicating. Using automation, a large number of cells can be scanned. In this proposal, we will employ this novelmethodtofirstquantitateandcharacterizelatencyincelllinemodels,thenthelatentreservoirsinbrain derivedmicrogliaandastrocytesanddelineatetheeffectofthreedrugsofabuse(methamphetamine,cocaine and cannabis) on the efficiency of formation of latent cells, the rate of reactivation of latent cells, and establishmentoflatencyacrossblood-brain-barrier(BBB).ThreeAimsareproposed.InAim1,wewillstudy theroleofillicitdrugsonthereactivationoflatentlyinfectedCNS-derivedcellsbyestablishinginvitroandex vivo models of latency. First, we will employ immortalized human monocyte and microglial cell lines to study the kinetics of reactivation and the effect of METH, Cocaine and cannabis on the reactivation using SMIRA. ThiswillbefollowedbysimilarstudiesusingaprimaryCNScells.InAim2,wewillstudytheroleofillicitdrugs ontheestablishmentofHIVreservoirsinCNScellsacrossBBB.UsingSMIRAtodetectHIV-1+cells,wewill determine the relative effect of drugs of abuse, antiretrovirals and the addition of LRAs to establish latency acrossBBB.InAim3,wewillstudytheeffectofillicitdrugsonCNSHIVreservoirsinHIV-infectedindividuals. Employing brain autopsy samples from HIV+ individuals with or without illicit drug use history (from the ManhattanBrainBank(NNTC),wewillcharacterizetherelativefrequencyofmicroglialandastrocytelatentcell reservoirs in which HIV-1 is actively replicating vs. those that are latent, using SMIRA and state-of-the-art, high-speed/resolutionwholeslidescanner,thePerkinElmerPannoramic250FlashII.Wewillstudytheeffect of drug abuse on the rates of reactivation. The proposed studies will provide valuable tools to study establishmentandreactivationofCNSlatencyinresponsetoillicitdrugsandprovidesadrugscreeningassay toeradicatetheselatentcellsinthefuture.